U.S. patent number 8,809,386 [Application Number 13/321,415] was granted by the patent office on 2014-08-19 for use of dynamin ring stabilizers.
This patent grant is currently assigned to Children's Medical Research Institute, The General Hospital Corporation. The grantee listed for this patent is Phillip J. Robinson, Sanja Sever. Invention is credited to Phillip J. Robinson, Sanja Sever.
United States Patent |
8,809,386 |
Robinson , et al. |
August 19, 2014 |
Use of dynamin ring stabilizers
Abstract
There is provided a method for promoting dynamin ring formation
and/or maintenance of dynamin rings in a cell, comprising treating
the cell with an effective amount of a dynamin ring stabilizer, or
a prodrug or pharmaceutically acceptable salt of the dynamin ring
stabilizer. The maintenance or accumulation of dynamin ring
formation has particular application in the prophylaxis or
treatment of a kidney disease or condition characterized by
proteinuria. A dynamin ring stabilizer can be any agent that
interacts with dynamin to promote dynamin ring assembly and/or
inhibit dynamin ring disassembly. There are also provided methods
for prophylaxis or treatment of podocyte dysfunction and/or
maintaining or inducing actin cytoskeleton formation in a cell
utilizing dynamin ring stabilizers, and for screening a test agent
for use as a dynamin ring stabilizer.
Inventors: |
Robinson; Phillip J. (North
Rocks, AU), Sever; Sanja (Brookline, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Robinson; Phillip J.
Sever; Sanja |
North Rocks
Brookline |
N/A
MA |
AU
US |
|
|
Assignee: |
Children's Medical Research
Institute (Westmead, NSW, AU)
The General Hospital Corporation (Boston, MA)
|
Family
ID: |
43125677 |
Appl.
No.: |
13/321,415 |
Filed: |
May 21, 2010 |
PCT
Filed: |
May 21, 2010 |
PCT No.: |
PCT/AU2010/000677 |
371(c)(1),(2),(4) Date: |
January 25, 2012 |
PCT
Pub. No.: |
WO2010/132959 |
PCT
Pub. Date: |
November 25, 2010 |
Prior Publication Data
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|
Document
Identifier |
Publication Date |
|
US 20120122968 A1 |
May 17, 2012 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61180261 |
May 21, 2009 |
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Current U.S.
Class: |
514/456 |
Current CPC
Class: |
A61K
31/275 (20130101); A61P 13/12 (20180101); A61P
29/00 (20180101); A61K 31/404 (20130101); A61P
9/12 (20180101); A61P 43/00 (20180101); A61K
31/352 (20130101); A61P 13/02 (20180101); A61P
37/06 (20180101) |
Current International
Class: |
A61K
31/35 (20060101) |
Field of
Search: |
;514/456 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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95/14464 |
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Jun 1995 |
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WO |
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01/64880 |
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Sep 2001 |
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WO |
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2005/049009 |
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Jun 2005 |
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WO |
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2007/056435 |
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May 2007 |
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WO |
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2009/034464 |
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Mar 2009 |
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WO |
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|
Primary Examiner: Weddington; Kevin E
Attorney, Agent or Firm: Rothwell, Figg, Ernst & Manbeck
P.C.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a national stage filing under 35 U.S.C.
.sctn.371 of PCT/AU2010/000677, filed on 21 May 2010 which in turn
claims priority of U.S. Provisional Application No. 61/180,261,
filed on 21 May 2009. The disclosures of each of the above
applications are hereby incorporated by reference in their
entireties into the present application.
Claims
The invention claimed is:
1. A method for prophylaxis or treatment of a kidney disease or
condition characterized by proteinuria, comprising administering to
an individual in need thereof an effective amount of at least one
dynamin ring stabilizer for maintaining and/or inducing actin
cytoskeleton formation, or prodrug or pharmaceutically acceptable
salt of the dynamin ring stabilizer.
2. A method according to claim 1 wherein the dynamin ring
stabilizer is a helical dynamin GTPase inhibitor.
3. A method according to claim 1 wherein the dynamin ring
stabilizer is not an inhibitor of dynamin GTPase activity.
4. A method according claim 1 wherein the dynamin ring stabilizer
is a compound that promotes formation of dynamin rings.
5. A method according to claim 1 wherein the dynamin ring
stabilizer is a compound that inhibits dynamin ring
disassembly.
6. A method according to claim 1 wherein the dynamin ring
stabilizer is selected from the group consisting of helical dynamin
GTPase inhibitors, dimeric tyrphostins, dimeric
benzylidenemalonitrile tyrphostins, iminochromenes, monomeric
tyrphostins, and 3-substituted naphthalene-2-carboxylic acid
(benzylidene) hydrazides.
7. A method according to claim 1 wherein the kidney disease or
condition is selected from the group consisting of nephrotic
syndrome, chronic kidney disease, glomerular disease, glomerular
dysfunction, glomerulonephritis, nephropathy, diabetic nephropathy,
podocyte dysfunction, podocyte injury, podocytopathies, podocyte
foot process effacement, diffuse mesengial sclerosis, congenital
nephrotic syndrome, Alpor's syndrome and variants, minimal change
disease, focal segmental glomerulosclerosis (FSGS), collapsing
glomerulonephropathy, immune and inflammatory
glomerulonephropathies, hypertensive nephropathy, and age
associated glomerulonephropathy.
8. A method according to claim 7 wherein the kidney disease or
condition is selected from the group consisting of nephrotic
syndrome, chronic kidney disease, glomerular disease, podocyte
dysfunction, and podocyte foot process effacement.
9. A method according to claim 8 wherein the kidney disease or
condition is podocyte foot process effacement.
10. A method according to claim 1 wherein the kidney disease or
condition comprises podocyte dysfunction characterized by podocyte
foot process effacement.
11. A method according to claim 1 comprising treating podocytes
with the dynamin ring stabilizer to maintain and/or induce
formation of podocyte foot processes.
12. A method according to claim 1 wherein the induction of actin
cytoskeleton formation comprises inducing formation of actin stress
fibres.
13. A method according to claim 1 comprising administering the
dynamin ring stabilizer for inducing focal adhesions and/or
formation of actin stress fibres in podocytes.
14. A method for prophylaxis or treatment of podocyte foot process
effacement, comprising administering to an individual in need
thereof an effective amount of at least one dynamin ring
stabilizer, or prodrug or pharmaceutically acceptable salt of the
dynamin ring stabilizer.
15. A method according to claim 14 comprising administering to the
individual an effective amount of the dynamin ring stabilizer or a
pharmaceutically acceptable salt of the dynamin ring
stabilizer.
16. A method according to claim 14 wherein the dynamin ring
stabiliser is selected from the group consisting of dimeric
tyrphostins, iminochromenes, monomeric typhostins, and
3-substituted naphthalene-2-carboxylic acid (benzylidene)
hydrazides.
17. A method according to claim 14 wherein the dynamin ring
stabiliser is a compound for promoting oligomerization of dynamin
into dynamin rings.
Description
SEQUENCE SUBMISSION
The present application includes a Sequence Listing filed in
electronic format. The Sequence Listing is entitled
3587103SequenceListingRevised.txt, was created on 25 Jan. 2012 and
is 9 kb in size. The information in the electronic format of the
Sequence Listing is part of the present application and is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to the use of a class of agents
referred to herein as "dynamin ring stabilizers" to promote
formation of, and/or maintain, stable dynamin rings in cells. The
invention has particular application in the prophylaxis or
treatment of kidney diseases or conditions characterized by
proteinuria.
BACKGROUND OF THE INVENTION
The global epidemic of chronic kidney disease (CKD) is progressing
at an alarming rate. Up to 11% of the general population is
affected in the US, Australia, Japan and Europe. There is a
simultaneous steady increase of type II diabetes, and its
associated kidney complications, particularly in India, China and
South-East Asia, and kidney-related diseases are eluding present
treatment options and resources. Histological and genetic data
strongly implicate podocyte dysfunction in glomerular disease
(Susztak and Bottinger 2006; Tryggvason et al. 2006).
One of the earliest events marking podocyte dysfunction is
disruption of foot processes (FP) and slit diaphragms, which is
thought to cause foot-process fusion and proteinuria (Susztak and
Bottinger 2006). In most cases of CKD, the first clinical sign is
proteinuria. If these early structural changes within podocytes are
not reversed, progressive, severe damage occurs, leading to
detachment of podocytes from the glomerular basement membrane
(GBM). This results in scarring, obliteration of the urinary space,
and development of segmental glomerulosclerosis and end stage renal
failure. Rearrangement of the actin cytoskeleton which links the
slit diaphragm, apical domain and sole plate, serves as a common
denominator during foot process (FP) effacement. Thus, a better
understanding of mechanisms controlling foot process formation in
health and disease is essential to design better early diagnostics
and therapies that intervene, while permanent damage may still be
preventable.
Renal filtration occurs in the glomerulus, a specialized structure
that ensures selectivity of the kidney filter so that water,
electrolytes and waste products are passed into the urinary space,
while essential plasma proteins are retained in the blood. A sign
of glomerular dysfunction is the loss of protein in the urine
termed proteinuria or nephrotic syndrome (defined as protein loss
exceeding 3.5 grams/day). Proteinuria often leads to progressive
renal failure, eventually requiring dialysis or kidney
transplantation. Together with the GBM and the glomerular
endothelial cells, podocytes form a key component of the kidney
permeability barrier. Podocyte function depends on a complex
cellular structure, which consists of a cell body, as well as major
processes and foot processes (FP) as described above. The FPs of
one podocyte are inter-digitated with those of its neighbors, and
the intercellular space between adjacent foot processes is bridged
by a slit diaphragm composed of the protein nephrin, which also
represents the final barrier to protein loss. Thus, podocyte injury
is tightly correlated with proteinuria.
Podocyte FPs contain an elaborate and dynamic actin-based
cytoskeleton that is essential for their membrane morphogenesis and
for establishing and maintaining the filtration barrier in the
kidney (Faul et al. 2007). FPs contain a microfilament-based
contractile apparatus composed of actin, myosin II,
.alpha.-actinin, talin, and vinculin, which is linked to the GBM at
focal contacts by an integrin .alpha.3.beta.1 complex (Faul et al.
2007). The FP actin is organized in two principle forms: a
podosome-like, cortical network of short branched actin filaments,
and stress fibers composed of an actin:myosin core occupying the
center of the FP (Ichimura et al. 2003). FP structure appears to be
optimized for constant actin-driven morphological rearrangements,
which are essential for glomerular filtration (Moeller and Holzman
2006).
Most forms of proteinuria and nephrotic syndrome involve a
reduction of podocyte membrane extensions and transformation of
podocyte FPs into a band of cytoplasm (i.e., FP effacement).
Changes in FP morphology are primarily driven by reorganization of
the actin cytoskeleton, which condenses into a thick bundle against
the sole of podocyte foot processes. A number of proteins directly
or indirectly alter podocyte cytoskeletal organization. For
example, mutations in .alpha.-actinin-4, which cause a late-onset
form of focal segmental glomerulosclerosis (FSGS), revealed the
importance of structural actin binding proteins for podocyte
function (Kaplan et al. 2000). Signals that originate at the slit
diaphragm can directly influence the actin cytoskeleton in
podocytes (Jones et al. 2006; Moeller et al. 2004).
It has been reported that cell focal adhesion turnover is mediated
through dynamin-clathrin-dependent endocytosis of activated .beta.1
integrins, and that knockdown of either dynamin II or both clathrin
adaptors AP-2 and disabled-2 (DAB2) blocks .beta.1 integrin
internalization leading to impaired focal adhesion disassembly and
cell migration (Chao and Kunz, 2009).
FP effacement during nephrotic syndrome is a migratory event
(Reiser et al. 2004). Cultured podocytes contain all three major
categories of actin structures required for cell migration:
lamellipodia, filopodia and contractile actin stress fibers.
Cultured podocytes also express all known differentiation markers
characteristic of podocytes including: nephrin, podocin, CD2AP,
synaptopodin, as well as known components of the slit diaphragm
such as ZO-1, P-cadherin, .alpha.-, .beta.-, and .gamma.-catenin
(Mundel et al. 1997; Saleem et al. 2002). Indeed, podocytes have
been extensively used to study known actin binding and bundling
proteins (e.g., .alpha.-actinin 4 and synaptopodin (Asanuma et al.
2005; Asanuma et al. 2006). The cortical actin web that underlies
formation of lamellipodia and filopodia in cultured podocytes
appears to be equivalent to the short-branched actin web in the
vicinity of the plasma membrane observed by EM in podocytes in vivo
(Ichimura et al. 2003). Similarly, actin-myosin stress fibers
observed in cultured podocytes are likely to be equivalent to
non-branched stress fibers occupying the center of FP in vivo
(Ichimura et al. 2003).
Cytoskeletal dynamics are often controlled by the Rho family of
small GTPases. At the leading edge of cells, Rac1 and Cdc42 promote
cell motility through the formation of cortical actin, which in
turn promotes motility through the formation of lamellipodia and
filopodia, respectively. In contrast, RhoA promotes the formation
of contractile actin-myosin-containing stress fibers in the cell
body. RhoA signaling plays an important role in regulating the
actin cytoskeleton in podocytes. Thus, synaptopodin, an
actin-binding protein expressed in podocytes (Mundel et al. 1997)
induces stress-fiber formation by extending the lifetime of active
RhoA (Asanuma et al. 2006). The exact roles of Rac1 and Cdc42
signaling for podocyte structure and function are less well
understood.
It has been reported that in some mouse models of nephrotic
syndrome, preservation of dynamin is sufficient to counteract early
stages of foot processes effacement and proteinuria (Sever et al.
2007). Dynamin is a large GTPase enzyme that severs membrane-bound
clathrin-coated vesicles. The clathrin-mediated endocytic pathway
is of special interest to biomedical researchers because it is
involved in internalizing activated receptors, sequestering growth
factors, antigen presentation, cytokinesis, synaptic transmission
and as an entry route for a variety of pathogens. Dynamin comprises
three major isoforms: dynamin I (neurons); dynamin II (ubiquitous)
and dynamin III (neurons and testes) (Cousin and Robinson 2001).
Common to all are five domains, a GTPase domain (required for
vesicle fission), a middle domain (MD, of unknown function),
pleckstrin homology domain (PH, targeting domain and potentially a
GTPase inhibitory module), a GTPase effector domain (GED, which
controls dynamin self-assembly into rings), and a proline-rich
domain (PRD, which interacts with proteins containing an SH3 domain
and is the main site for dynamin I and III phosphorylation in
vivo).
Dynamin is best known for its roles in clathrin-mediated
endocytosis at the plasma membrane and synaptic vesicle endocytosis
in neurons (Sever et al. 2000b). A number of studies indicate that
dynamin has additional roles, including regulation of the actin
cytoskeleton through molecular mechanisms that are poorly
understood (Schafer 2004). Dynamin's role in regulation of the
actin cytoskeleton has been attributed to its interactions with
known actin binding and regulatory proteins such as profilin, Nck
and cortactin (Orth and McNiven 2003; Schafer 2004). A previous
study has also indicated that dynamin is essential for formation of
functional FPs in podocytes (Sever et al. 2007).
Dynamin exhibits unique biochemical characteristics distinct from
other GTPases, such as high molecular weight (MW=100 kDa) and
atypically low affinity for GTP (K.sub.m=.about.10 .mu.M). Dynamin
exists in three main states--basal, ring or helix--and its GTPase
activity increases stepwise upon transition to each state. More
particularly:
a) In its "basal" state dynamin is in equilibrium between monomer,
dimers and homotetramers (Muhlberg et al. 1997), and has a "basal"
GTPase rate of .about.1 min.sup.-1.
b) Dynamin dimers or tetramers can further self-assemble into
higher-order structures resembling "rings" that have an outer
diameter of about 50 nm and an inner opening of about 30 nm
(Hinshaw and Schmid, 1995). This typically occurs above 500 nM
dynamin in vitro. The rings are not always closed and the diameter
can vary between systems. Ring formation is promoted by dialysis of
dynamin into low salt buffers and occurs with high concentrations
of dynamin of around 0.5-1 micromolar. Ring formation stimulates
dynamin's GTPase activity about 10 fold (Warnock et al. 1996). The
increase in the rate of GTP hydrolysis is due to activation of
intramolecular GTPase activating domain within dynamin that only
becomes active when dynamin tetramers come together (Sever et al.
1999). A dynamin mutant has been reported that is predicted to live
longer in the ring form--dynR725A is a mutant impaired in
stimulated rate of GTP hydrolysis (Sever et al. 2000a).
c) In the presence of an assembly template dynamin can further
assemble into a "helix" in vitro. The helix assembly templates
include phospholipid liposomes, lipid nanotubes or microtubules.
The helix appears to be an extension of the individual ring
structure into a highly elongated helical structure, much like a
spring. Helix formation stimulates dynamin's GTPase activity
100-1000 fold (Warnock et al. 1996). The stimulated rate of GTP
hydrolysis in turn drives dynamin disassembly in vitro, and leads
to loss of positive cooperativity for GTP-binding (Sever et al.
2006).
There is an emerging new field of dynamin pharmacology with the
development of small-molecule inhibitors specific for the dynamin
family of GTPases as powerful new tools with which to study
cellular endocytosis in these systems. Small molecule dynamin
inhibitors have attracted widespread attention and have been used
to study endocytosis and other aspects of membrane dynamics in a
variety of cellular systems (Macia et al. 2006). These inhibitors
offer many distinct advantages over traditional means of dynamin
inhibition in cells by expression of dynamin GTPase mutants or by
small interfering RNA (siRNA)-mediated dynamin knockdown which
cannot be used to study rapid cellular effects. Small molecule,
cell-permeable inhibitors are able to rapidly block endocytosis in
minutes and are readily reversible (Macia et al. 2006; Quan et al.
2007).
The first reported dynamin inhibitors were long chain ammonium
salts such as myristyl trimethyl ammonium bromide (MiTMAB),
octadecyltrimethyl ammonium bromide (OcTMAB) (Hill et al. 2004) and
dimeric tyrphostins such as Bis-T (Hill et al. 2005). Later a
series of room temperature ionic liquids (RTILs) (Zhang et al.
2008) and dynasore (Macia et al. 2006) were reported. Finally,
indole-based inhibitors termed "dynoles" (Hill et al. 2009) and
iminochromene-based inhibitors termed "iminodyns" have been
reported (Hill et al. 2010). Most studies screening for dynamin
inhibitors use GTPase assays whereby dynamin is maximally
stimulated, and likely to be in its helical state. Some of the most
potent inhibitors from each of these series are also potent and
reversible inhibitors of endocytosis in cells (Quan et al. 2007;
Hill et al. 2009; Hill et al 2010).
SUMMARY OF THE INVENTION
Broadly, the invention stems from two discoveries. Firstly, it has
been found that a subgroup of dynamin modulators can promote the
accumulation of dynamin in its oligomerised ring state and retard
dynamin ring disassembly. The compounds in this subgroup are termed
"dynamin ring stabilizers". One consequence of prolonging dynamin
ring lifetime is that this stimulates basal dynamin GTPase
activity, another is that this facilitates the formation of
filamentous actin (F-actin). Secondly, it has been found that
dynamin directly binds actin via the dynamin middle domain (MD),
promoting its oligomerization into rings which have a direct role
in de novo formation of focal adhesions and actin filaments in
podocytes. Stimulation of dynamin rings (as distinct from dynamin
helices) is a new cellular function for dynamin that is separate
from its known endocytic role. Prolonging dynamin ring formation
and/or lifetime has particular application in the prophylaxis or
treatment of foot process effacement in podocytes and proteinuric
kidney diseases.
In an aspect of the invention there is provided a method for
promoting dynamin ring formation and/or maintenance of dynamin
rings in a cell, comprising treating the cell with an effective
amount of a dynamin ring stabilizer, or a prodrug or
pharmaceutically acceptable salt of the dynamin ring
stabilizer.
In another aspect of the invention there is provided a method for
prophylaxis or treatment of a kidney disease or condition
characterized by proteinuria, comprising administering to an
individual in need thereof an effective amount of at least one
dynamin ring stabilizer, or a prodrug or pharmaceutically
acceptable salt of the dynamin ring stabilizer.
In another aspect of the invention there is provided a method for
prophylaxis or treatment of podocyte dysfunction, comprising
treating the podocyte with an effective amount of at least one
dynamin ring stabilizer, or a prodrug or pharmaceutically
acceptable salt of the dynamin ring stabilizer.
Typically, the podocyte dysfunction is characterized by, or is
associated with, foot process effacement.
In another aspect of the invention there is provided a method for
maintaining or inducing actin cytoskeleton formation in a cell,
comprising treating the cell with an effective amount of at least
one dynamin ring stabilizer, or a prodrug or pharmaceutically
acceptable salt of the dyamin ring stabilizer.
In another aspect of the invention there is provided a method for
inducing focal adhesions and/or actin stress fibres in a cell,
comprising treating the cell with an effective amount of at least
one dynamin ring stabilizer, or a prodrug or pharmaceutically
acceptable salt of the dyamin ring stabilizer.
In another aspect there is provided a method of screening a test
agent for use as a dynamin ring stabilizer, comprising:
providing the test agent;
incubating the test agent with dynamin protein under conditions
suitable for the formation of dynamin rings; and
evaluating whether the test agent promotes accumulation of dynamin
rings and/or inhibits disassembly of dynamin rings, the
accumulation of dynamin rings and/or inhibition of disassembly of
dynamin rings increasing basal dynamin GTPase activity.
The evaluation of whether the test agent promotes the accumulation
of dynamin rings or inhibits disassembly of dynamin rings can
involve assaying for an increase in basal dynamin GTPase activity,
and/or release of dynamin that is indicative of dynamin ring
disassembly.
In another aspect of the invention there is provided a dynamin ring
stabilizer for use in promoting dynamin ring formation and/or
maintenance of dynamin rings in a cell, or a prodrug or
pharmaceutically acceptable salt of the dynamin ring
stabilizer.
In another aspect of the invention there is provided at least one
dynamin ring stabilizer for use in the prophylaxis or treatment of
a kidney disease or condition characterized by proteinuria, or a
prodrug or pharmaceutically acceptable salt of the dynamin ring
stabilizer.
In another aspect of the invention there is provided the use of at
least one dynamin ring stabilizer in the manufacture of a
medicament for promoting dynamin ring formation and/or maintenance
of dynamin rings in cells of an individual in need thereof, or a
prodrug or pharmaceutically acceptable salt of the dynamin ring
stabilizer.
In still another aspect of the invention there is provided the use
of at least one dynamin ring stabilizer in the manufacture of a
medicament for prophylaxis or treatment of a kidney disease or
condition characterized by proteinuria, or a prodrug or
pharmaceutically acceptable salt of the dynamin ring
stabilizer.
The dynamin ring stabilizer used in an embodiment of the invention
can be any such compound that promotes assembly, or inhibits
disassembly, of dynamin rings. The inhibition can be retardation or
prevention of dynamin ring disassembly.
By the term "dynamin ring" as used herein is meant a ring of
oligomerised dynamin units. The ring can be a closed ring or a
single turn of a dynamin helix (helical dynamin).
By the term "dynamin ring stabilizer" as used herein is meant an
agent that interacts with dynamin and stimulates basal dynamin
GTPase activity in the absence of an assembly template (e.g.,
microtubules, phospholipid vesicles and/or nanotubes) around which
dynamin helices form. A dynamin ring stabilizer promotes dynamin
ring assembly and/or inhibits dynamin ring disassembly, both of
which may result in dynamin ring accumulation and/or an increase in
basal dynamin GTPase activity. Hence, an agent that promotes
dynamin ring assembly and/or inhibits dynamin ring disassembly is
encompassed by the term "dynamin ring stabilizer" in the context of
the present invention. Typically, the dynamin ring stabilizer will
be an agent that inhibits dynamin ring disassembly.
The stimulation of basal dynamin GTPase activity by the dynamin
ring stabilizer is to a level less than that associated with
maximally active helical dynamin whereby maximal activity is
achieved in the presence of an assembly template.
The interaction of the dynamin ring stabilizer with dynamin can be
binding of the dynamin ring stabilizer to dynamin, or by direct or
indirect association of the dynamin ring stabilizer with dynamin.
When dynamin is in its helical state, the dynamin ring stabilizer
may increase the GTPase activity of individual dynamin rings within
that helix, but to a level lower than that achieved by co-operative
interaction between dynamin rings.
Most typically, the dynamin ring stabilizer utilized in a method
embodied by the invention is an inhibitor of the GTPase activity of
maximally stimulated helical dynamin. Likewise, the test agent
screened for use as a dynamin ring stabilizer can be an inhibitor
of the GTPase activity of helical dynamin. However, from the above
it will be understood that the dynamin ring stabilizer need not be
an inhibitor of dynamin ring disassembly and indeed, need not be an
inhibitor of helical dynamin GTPase activity.
The dynamin with which the dynamin ring stabilizer interacts and/or
the dynamin from which the dynamin ring or rings are formed, can be
selected from the group consisting of dynamin I (dynI), dynamin II
(dynII), dynamin III (dynIII), and dynamin isoforms, and mixtures
of the foregoing.
The features and advantages of invention will become further
apparent from the following detailed description of non-limiting
embodiments together with the accompanying drawings.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
FIG. 1 is a graph showing that the dimeric benzylidenemalonitrile
tyrphostin Bis-T-23 inhibits lipid-stimulated dynamin, but
stimulates basal dynamin GTPase activity.
FIG. 2 is a graph showing inhibition and activation of dynamin
occur at the same Bis-T-23 concentration.
FIG. 3 is a graph showing Bis-T-23 (5 .mu.M) stimulation of basal
dynamin GTPase activity occurs after a "lag phase".
FIG. 4 is a graph showing the relative potency of three iminodyns
on stimulating the basal GTPase activity of full length dynamin I
(200 nM, no other known activators such as PS liposomes were
present). The compounds were present at the indicated
concentrations and the concentration causing half-maximal
stimulation is shown as the EC.sub.50.
FIG. 5 is a graph showing the basal GTPase activity of the
truncated dynamin I construct known as GG2, containing only the
GTPase domain linked to a small part of the GED domain (Chappie et
al 2009). Unlike full length dynamin this construct is known to be
unable to self-assemble or to form rings. Bis-T-22 and -23 and
imminodyn-22 have no effect on the basal activity of this
assembly-incompetent construct.
FIG. 6 shows that Bis-T-22 at high concentration [dyn] (520 nM)
promotes dynamin ring self-assembly.
FIG. 7 shows that Bis-T-23 prevents dynamin disassembly.
FIGS. 8 (A) to (D) are electron micrographs showing Bis-T-23
prevents constriction or helical expansion of dynamin on PS
liposomes in vitro. It stabilizes the dynamin helix, which loses
its flexibility and forms uniform diameter rings, inhibiting
dynamin ring disassembly.
FIG. 9 shows the effect of treating purified full length dynamin I
with Bis-T-23 in the absence of a template. Dynamin was then
examined by electron microscopy. Dynamin rings are specifically
induced by Bis-T-23 (5.4 .mu.M, A). The rings are bona fide dynamin
rings (B). Quantitative analysis shows a 6-fold increase in bona
fide rings (C).
FIG. 10 shows that Bis-T-22 (100 .mu.M for 30 minutes) induces
clathrin coated pits in cells that have unusually long necks and
are encircled by rings in both cultured human lymphoblast cells (A)
or rat brain synaptosomes (B).
FIG. 11 (A) is a graph showing Bis-T-23 and two "dyngo" series
dynamin inhibitors are dynamin ring stabilizers. Dyngo-7a is also
known as dynasore. Dynamin I (50 nM) GTPase stimulation was
performed in the absence of the detergent Tween-80 (and in the
absence of any known activators such as PS liposomes, nanotubes or
microtubules). In contrast, dynole 34-2, a potent dynamin
inhibitor, does not stimulate basal dynamin activity under these
conditions. B) is a graph showing variety of potent dynamin
inhibitors are activators of basal dynamin GTPase activity (Bis-T
series compounds were used at a conc. of 10 .mu.M, all other
compounds were used at a conc. of 30 .mu.M). All of the compounds
tested inhibited phosphatidylserine (PS)-stimulated dynamin between
300 nM-3 .mu.M (data not shown). The GTPase activity of dynamin I
(dynI; 200 nM) was measured in the absence of lipid activators (and
in the presence of standard Tween-80 at 0.06%).
FIG. 12 shows detection of direct dynamin-actin interactions using
standard co-sedimentation assays in which F-actin sediments under
high-speed centrifugation.
FIG. 13 shows amino acid sequence alignments between dynamin II
(dyn2) (splice variants a and b) (SEQ ID No. 1 and SEQ ID No. 2),
dynamin I (dyn1) (splice variants a and b) (SEQ ID No. 3 and SEQ ID
No. 4), Drosophila (Shi) (SEQ ID No. 5), C. elegans (Cele) (SEQ ID
No. 6), yeast (Vps1) (SEQ ID No. 7). Dnm1 (SEQ ID No. 8), `loss of
function` mutants DynK/E (SEQ ID No. 9) and Dyn K/A (SEQ ID No.
10), and `gain of function` mutant Dyn E/K (SEQ ID No. 11). Dnm1 is
a dynamin family member involved in mitochondrial
morphogenesis.
FIG. 14 shows a Scatchard Plot of the direct dynamin and F-actin
interactions. Increasing concentrations of dynamin (free) were
added to 5 .mu.M of F-actin. After centrifugation at
100,000.times.g proteins were separated on SDS-PAGE and bands were
analyzed using densitometry.
FIGS. 15 (A-E) are photographs illustrating that dynamin-actin
interactions are essential for organization of the actin
cytoskeleton in podocytes. Podocytes were infected with viruses
expressing dynamin mutants as indicated. The actin cytoskeleton
appears greatly increased in the cells expressing dynE/K and
dynR725A.
FIG. 16 shows assembled dynamin rings cross-link actin filaments
into thin bundles in the presence of GTP.gamma.S. Actin filaments
were visualized using negative staining and electron microscopy.
Arrows point toward dynamin rings along the bundled filaments.
FIG. 17 is a graph showing dynamin rings promote actin
polymerization. Representative time courses of the repolymerization
of actin when 0.33 .mu.M Gsn-actin complexes (G1:200A or G1:1000A)
are incubated in the absence and presence of 0.1 .mu.M dynamin with
or without 100 .mu.M GTP.gamma.S.
FIG. 18 is a graph showing the dimeric benzylidenemalonitrile
tyrphostin Bis-T-22 stimulates dynamin's basal rate of GTP
hydrolysis. Shot gelsolin-capped F-actin also stimulates dynamin's
basal rate of GTP hydrolysis. Time course for basal GTP hydrolysis
of 0.2 .mu.M dyn1 incubated without or with 7 .mu.M Bis-T-22, and
with or without 10 .mu.M Gsn-F-actin complexes. The effects of
Bis-T-22 and Gsn-F-actin are at least additive.
FIG. 19 is a photograph showing the effects of Bis-T-23 on the
actin cytoskeleton in podocytes. Cells were stained using
rhodamine-phalloidin for F-actin (left column) and anti-paxillin
monoclonal antibody (centre column). The merged staining is shown
on the right column. The actin cytoskeleton and focal adhesions are
greatly increased in number and amount in the cells treated with
Bis-T-23 and cells expressing dynamin mutants, dynE/K and
dynR725A.
FIG. 20 is a graph showing a control mouse and a mouse expressing
`gain of function` .alpha.-actinin 4 mutant protein were injected
intraperitoneally with Bis-T-23 (100 .mu.g/100 g body weight).
Proteinuria was measured using mouse Albumin-specific ELISA and
Creatinine Companion assay kits (Exocell and Bethyl Laboratories)
following manufacturer's protocols. A decrease in proteinuria to
wild-type levels was obtained up to 6 hours after drug
administration.
FIG. 21 is a graph showing that the ring stabilizer Bis-T-23
rescues proteinuria in lipopolysaccharide (LPS) treated mice. LPS
is a model for proteinuric kidney disease. Albumin levels were
determined in 6 mice prior to (Con), 24 hours after LPS injection
(2.times.LPS), and 2, 4, 6 and 8 h after administration of Bis-T-23
(open bars) or DMSO (delivery solution, grey bars). A decrease in
proteinuria was noted from 2-8 hours after administration of a
single dose of this ring stabilizer.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION
A subgroup of dual-specificity dynamin modulators has been found to
exist among the broader group of dynamin inhibitors. The compounds
in this subgroup are "dynamin ring stabilizers" as they inhibit
dynamin ring disassembly thereby prolonging dynamin ring lifetime
and promoting dynamin ring accumulation. This is consistent with
the fact that GTP hydrolysis is known to drive dynamin disassembly
(Sever et al. 2006). However, while these compounds reduce the
massive GTPase activity of helical dynamin, they can simultaneously
increase the basal GTPase activity of individual dynamin rings.
A dynamin ring is a single turn of oligomerized dynamin or in the
case of helical dynamin (a dynamin helix), a single turn of the
helix. Dynamin rings were first observed in vitro (Hinshaw and
Schmid. 1995). They typically have an outer diameter of
approximately 50 nm and an inner diameter of about 30 nm, and the
rings can be open or closed. Helical dynamin is also known as a
dynamin helix, nano-spring, spiral or "stack of rings" (Stowell et
al. 1999). Cryo-electron microscopy indicates that dynamin ring
size is flexible and can comprise 13-15 asymmetric repeated dynamin
units, suggesting that a single ring of helical dynamin comprises
26-30 dynamin molecules (dynamin units) (Zhang and Hinshaw. 2001).
However, since the ring diameter is flexible, these numbers are not
fixed.
A dynamin ring stabilizer useful in a method embodied by the
invention may for instance be selected from the group consisting of
helical dynamin GTPase inhibitors, monomeric tyrphostins, dimeric
tyrphostins and particularly dimeric benzylidenemalonitrile
tyrphostins, iminochromenes, 3-substituted naphthalene-2-carboxylic
acid (benzylidene) hydrazides, polypeptides and peptides as further
described below.
Suitable dimeric benzylidenemalonitrile tyrphostins (Bis-T) and
related compounds that may find application as dynamin ring
stabilizers in accordance with embodiments of the invention are
described in International Patent Application No. PCT/AU2004/001624
(WO 2005/049009) and Hill et al. 2005, the contents of which are
incorporated herein in their entirety.
Bis-tyrphostin-22 (Bis-T-22) is one such dimeric typhostin and is a
potent in vitro inhibitor of dynamin when dynamin is activated by
phosphatidylserine (PS) liposomes to assemble into a flexible
helix. In the absence of PS liposomes, dynamin can only assemble
into single rings. Surprisingly, while Bis-T-22 inhibits the
activity of helical dynamin it also uniquely, simultaneously
stimulates basal dynamin GTPase activity by preventing disassembly
of dynamin rings. The structure of Bis-T-22 is shown below. Bis-T
has the same structure as Bis-T-22 but has an additional hydroxyl
substituent on the C5 carbon atom of each terminal phenyl
group.
##STR00001##
Particularly suitable dimeric tyrphostins useful as dynamin ring
stabilizers include those Bis-T compounds in which two of the C3-C5
carbon atoms of at least one terminal phenyl ring have hydroxyl
(OH) substituents, preferably in a catechol arrangement (e.g., as
in Bis-T-22), or all three of the carbon atoms are substituted with
hydroxyl (e.g., as in Bis-T-23). Examples include
2-cyano-N-{3-[2-cyano-3-(3,4-dihydroxyphenyl)-acryloylamino]-ethyl}-3-(3,-
4-dihydroxyphenyl)-acrylamide,
2-cyano-N-{3-[2-cyano-3-(3,4,5-trihydroxyphenyl)-acryloylamino]-ethyl}-3--
(3,4,5-trihydroxyphenyl)-acrylamide,
2-cyano-N-{3-[2-cyano-3-(3,4-dihydroxy-4-methoxyphenyl)-acryloylamino]-et-
hyl}-3-(3,4-dihydroxy-5-methoxyphenyl)-acrylamide,
2-cyano-N-{3-[2-cyano-3-(3,4-dihydroxyphenyl)-acryloylamino]-propyl}-3-(3-
,4-dihydroxyphenyl)-acrylamide (Bis-T-22),
2-cyano-N-{3-[2-cyano-3-(3,4,5-trihydroxyphenyl)-acryloylamino]-propyl}-3-
-(3,4,5-trihydroxyphenyl)-acrylamide (Bis-T-23),
2-cyano-N-{3-[2-cyano-3-(3,4-dihydroxy-5-methoxyphenyl)-acryloylamino]-pr-
opyl}-3-(3,4-dihydroxy-5-methoxyphenyl)-acrylamide,
2-cyano-N-{3-[2-cyano-3-(3,4-dihydroxyphenyl)-acryloylamino]-butyl}-3-(3,-
4-dihydroxyphenyl)-acrylamide,
2-cyano-N-{3-[2-cyano-3-(3,4,5-trihydroxyphenyl)-acryloylamino]-butyl}-3--
(3,4,5-trihydroxyphenyl)-acrylamide,
2-cyano-N-{3-[2-cyano-3-(3,4-dihydroxy-5-methoxyphenyl)-acryloylamino]-bu-
tyl}-3-(3,4-dihydroxy-5-methoxyphenyl)-acrylamide,
2-cyano-N-{3-[2-cyano-3-(3,4-dihydroxyphenyl)-acryloylamino]-pentyl}-3-(3-
,4-dihydroxyphenyl)-acrylamide,
2-cyano-N-{3-[2-cyano-3-(3,4,5-trihydroxyphenyl)-acryloylamino]-pentyl}-3-
-(3,4,5-trihydroxyphenyl)-acrylamide,
2-cyano-N-{3-[2-cyano-3-(3,4-dihydroxy-5-methoxyphenyl)-acryloylamino]-pe-
ntyl}-3-(3,4-dihydroxy-5-methoxyphenyl)-acrylamide,
2-cyano-N-{3-[2-cyano-3-(3,4-dihydroxyphenyl)-acryloylamino]-hexyl}-3-(3,-
4-dihydroxyphenyl)-acrylamide,
2-cyano-N-{3-[2-cyano-3-(3,4,5-trihydroxyphenyl)-acryloylamino]-hexyl}-3--
(3,4,5-trihydroxyphenyl)-acrylamide, and
2-cyano-N-{3-[2-cyano-3-(3,4-dihydroxy-5-methoxyphenyl)-acryloylamino]-he-
xyl}-3-(3,4-dihydroxy-5-methoxyphenyl)-acrylamide.
Further dynamin ring stabilizers include those in which a
substituent on the C2 carbon atom of at least one terminal phenyl
ring of a Bis-T compound and the position occupied by an adjacent
cyanyl group (CN) are cyclised as described in WO 2005/049009. For
instance, when the substituent is hydroxy, the hydroxy group can
react with the cyanyl group to form an iminochromene as
follows:
##STR00002##
where for example, R.sub.1 is OH, R.sub.2 is OH and R.sub.3 is H;
R.sub.1 is H, R.sub.2 is OH and R.sub.3 is OH; or R.sub.1, R.sub.2
and R.sub.3 are OH; and n is usually 0, 1, 2 or 3, and most usually
1. Further examples of iminochromenes useful in embodiments of the
invention are described below (see Table 2). Analogs of Bis-T or
iminochromene compounds as described above in which at least one of
the ring oxygen atoms and/or at least one of the NH groups and/or
backbone oxygen atoms of the compound are subjected to bioisostere
replacement may also be used (e.g., see Lima and Barreiro. 2005,
the contents of which is incorporated herein in its entirety by
cross-reference).
In addition, asymmetric analogues of the above dimeric compounds
may be utilized. Examples include the asymmetric azido and
diazarinyl analogs of dimeric tyrphostins described by Odell et al.
2009. Moreover, monomeric tyrphostin analogs of the dimeric
tyrphostins exemplified above can be utilized. However, in the case
of these tyrphostins, they are not GTPase inhibitors (e.g., see
Hill et al. 2005).
Yet further examples of dynamin ring stabilizers include
3-hydroxynaphthalene-2-carboxylic acid (3,4-dihydroxybenzylidene)
hydrazide (dynasore) and analogs thereof. The structure for
dynasore is as follows.
##STR00003##
Dynasore was discovered in a library screen using recombinant
dynamin I activated by the SH3 domain containing protein grb2
(Macia et al. 2006). The structure of dynasore is superficially
similar to that of Bis-T-22 where the position and number of the
hydroxyls around the terminal phenyl ring of Bis-T-22 was found to
contribute significantly to the dynamin inhibitory potency of the
compound. Examples of further 3-substituted
naphthalene-2-carboxylic acid (benzylidene) hydrazide analogs of
dynasore (named dyngo compounds herein) found to exhibit improved
dynamin inhibitory potency compared to dynasore are shown in Table
1. Each of the dyngo compounds was synthesized by a simple one-step
condensation reaction coupling 3-hydroxy-2-naphthoic acid hydrazide
with a variety of hydroxyl substituted benzaldehydes affording a
focussed library as illustrated by Scheme 1 below (e.g., by mixing
the reagents in ethanol (e.g., 10 ml) in a round bottomed flask,
refluxing the mixture for 2 hours, allowing it to cool and removing
the solvent in vacuo prior to recrystallizing the product from
ethanol although other synthesis methods available). In particular,
Table 1 shows the structure of each dyngo compound, its molecular
weight (MW) and IC.sub.50 for inhibition of native brain dynamin I
GTPase activity stimulated by PS liposomes in the presence or
absence of Tween 80.
##STR00004##
TABLE-US-00001 TABLE 1 Dyngo compounds MW Compound Structure
(g/mol) IC.sub.50 (.mu.M) IC.sub.50 (.mu.M) Dynasore (Dyngo-7a)
##STR00005## Full-length Dyn I (7 nM, 2 .mu.g/mol PS) With tween 80
Full-length Dyn I (7 nM, 2 .mu.g/ml PS) Without tween 80 Dyngo-4a
##STR00006## 338.32 2.7 .+-. 0.7 0.31 .+-. 0.05 Dyngo-6a
##STR00007## 322.31 19.8 22.1 Dyngo-1a ##STR00008## 322.31 200 7.3
Dyngo-5a ##STR00009## 338.32 ~468 4.4 Dyngo-2a ##STR00010## 352.35
1170 3.6 Dyngo-3a ##STR00011## 338.32 Not Active 3.1 Dyngo-8a
##STR00012## 322.31 Not Active 39.8
A dynamin ring stabilizer which stimulates the basal activity of
dynamin or inhibits disassembly of dynamin rings as described
herein will typically have a terminal phenyl group with hydroxyl
substituents on at least two of three consecutive carbon atoms of
the phenyl ring, as in Bis-T-22 and Bis-T-23. However, while the
terminal phenyl groups of the above exemplified dimeric tyrphostin,
iminochromene and dyngo analogs are substituted with hydroxyl
groups, persons of ordinary skill in the art will appreciate one or
more of those hydroxyls may be subjected to bioisosteric
replacement (such as but not limited to, an --NH.sub.2 group or a
halo atom such as F, Cl, Br or I, and the like). Likewise, a person
of ordinary skill in the art will also recognize that other changes
may be made to the dimeric benzylidenemalonitrile tryphostin,
iminochromene, monomeric tyrphostin, dynasore and dyngo compounds
described above such that dynamin ring stabilizing activity of the
compound is retained or enhanced, and any such analogs and modified
forms thereof can be used in a method as described herein. Examples
of modifications include, but are not limited to replacement of one
or more backbone ring carbon atoms for heteroatom(s) (e.g.,
independently selected from O, N and S) and/or other modifications
to those ring systems. The naphthalene group of the dyngo compounds
exemplified above is particularly amenable to such modifications
and/or bioisoteric replacement, and a large number of modified such
compounds useful in methods embodied by the invention are possible.
Such modifications and bioisosteric replacements as described above
are well within the scope of a person of ordinary skill in the art
(e.g., see Lima and Barreiro. 2005) and all are expressly
encompassed by the present invention. Indeed, any suitable
physiologically acceptable dynamin ring stabilizer can be employed.
Further suitable such compounds for use in herein may be identified
by screening chemical compound and combinatorial libraries, such
screening being well within the scope of the addressee.
Suitable iminochromenes (termed "iminodyns" herein) and related
compounds that may find application as dynamin ring stabilizers in
accordance with embodiments of the invention are described in Hill
et al. 2010, the contents of which are incorporated herein in its
entirety by cross-reference.
Iminodyn-22 is one such iminochromene and is a potent in vitro
inhibitor of dynamin when dynamin is activated by
phosphatidylserine (PS) liposomes to assemble into a flexible
helix. While iminodyn-22 inhibits the activity of helical dynamin
it also simultaneously stimulates basal dynamin GTPase activity by
preventing disassembly of dynamin rings. The structure of
iminodyn-22 is shown below.
##STR00013## The pathway for synthesis of the iminodyns is shown
below in Scheme 2.
##STR00014##
The structure of iminochromene is distinct from that of Bis-T-22
but the position and number of the hydroxyls around the terminal
phenyl rings of contribute significantly to the dynamin inhibitory
potency of the compound. Examples of further analogs of
iminochromene are shown in Table 2. The table shows the structure
of each compound, its IC.sub.50 for inhibition of native brain
dynamin I (at 20 nM) GTPase activity stimulated by PS liposomes.
The table also shows the stimulation of basal dynamin I (at 200 nM)
GTPase activity in the absence of liposomes.
TABLE-US-00002 TABLE 2 Inhibition of dynamin I in vitro GTPase
activity, and ring stabilizer activity of iminodyns 17, and 20-23
PS-stimulated Dyn I Ring GTPase stabilizer: Iminodyn activity Dyn I
(no PS) compound Iminodyn structure IC.sub.50 (.mu.M) EC.sub.50
(.mu.M) 17 ##STR00015## 0.33 .+-. 0.07 31 20 ##STR00016## 36.6 .+-.
7.2 Not Active 21 ##STR00017## 17.3 .+-. 1.0 40 22 ##STR00018##
0.45 .+-. 0.05 1 23 ##STR00019## 0.26 .+-. 0.08 4
In another form, the dynamin ring stabilizer may be a peptide,
polypeptide or an active fragment or modified form thereof which
has dynamin ring stabilizing activity. Typically, a dynamin ring
stabilizer as described herein is other than a wild-type or
modified form of dynamin or a fragment thereof. Examples of
polypeptides or peptides that may be used include actin
(particularly F-actin), isoforms and/or fragments thereof that
provide an actin binding domain for one or more of the dynamin
isoforms or for dynamin rings, that promote/stimulate the formation
of dynamin rings and thereby act as a dynamin ring stabilizer in
the context of the invention. Further, modified forms may be
provided in which one or more amino acids are added, substituted or
deleted compared to the wild type actin, isoforms and fragments
thereof substantially without adversely impacting on its/their
capacity to interact with dynamin and promote the accumulation of
dynamin rings as described herein, and the use of all such modified
forms is also expressly encompassed.
Strategies for identifying such proteinaceous agents suitable for
use in methods of the present invention include large scale
screening techniques. For instance, phage display library protocols
provide an efficient way of screening a large number of potential
agents. The library utilised can be a peptide display library
expressing randomised peptide sequences fused to a coat protein of
the relevant phage utilised, or a library displaying variable
domains of antibodies (e.g., Fv fragments). Phages which bind to
dynamin can be recovered and amplified by infection of host
bacteria. Each clone isolated in this way expresses a specific
peptide or antigen-binding particle. The genes encoding the peptide
or antigen-binding particle are unique to each phage and can be
identified by recovering the DNA of the selected phage clone,
sequencing the DNA and comparing the sequence obtained with the
known sequence of the phage coat protein expressing the peptide or
antigen-binding molecule. The identified DNA can then be used for
expression of the encoded proteinaceous agent or modified to
provide other such agents utilising recombinant techniques well
known in the art.
A compound (whether a dynamin inhibitor or not) can be identified
as a dynamin ring stabilizer by assaying for its capacity to
promote accumulation of dynamin rings and/or to inhibit disassembly
of dynamin rings. This can primarily be determined by incubating
the test agent with full length dynamin under conditions in which
dynamin rings do not form of their own accord, and assaying for an
increase in basal dynamin GTPase activity to a level indicative of
the formation or accumulation of dynamin rings relative to
control(s). GTPase activity of dynamin or dynamin rings can be
determined by any conventionally known method (e.g., see Quan and
Robinson. 2005). Such conditions include: (a) the absence of a
helix assembly template such as liposomes, microtubules or lipid
nanotubes, and (b) higher concentrations of dynamin than normally
required for detecting template stimulated activity, typically
50-500 nM dynamin instead of 1-20 nM. Thus, ring stabilizer
activity can be identified by the ability to stimulate the GTPase
activity of full length dynamin in the absence of other stimulatory
factors (such as PS liposomes, microtubules or nanotubes).
Additional defining characteristics are available but are not
essential if the first condition has been met. One such additional
characteristic of ring stabilizer activity is that the stimulated
activity occurs after a lag phase of a few minutes, rather than
being an immediate activation in vitro. Another characteristic is
that a ring stabilizer cannot stimulate the activity of a dynamin
construct that is not capable of self-assembly, such as a mutant
dynamin or a construct containing the GTPase domain and only a
fragment of the GED domain (e.g., GG2 or GG5; Chappie et al 2009).
Yet another characteristic of ring stabilizer activity is the
ability of the compound to promote the formation of dynamin rings
as detected by electron microscopy at such concentrations of
dynamin whereby it does not form rings of its own accord. Such
conditions typically mean utilising 50-200 nM dynamin in the
absence of a template as distinct from higher concentrations where
self-assembly is known to occur without the addition of a dynamin
ring stabilizer.
An in-cell indicator of ring stabilizer activity is the induction
of actin stress fibres and focal adhesions after application of the
compound to cultured podocytes or NIH3T3 cells. The induction of
podocyte foot processes can also be assessed as an indicator of the
accumulation of dynamin rings and/or inhibition of dynamn ring
disassembly.
A peptide or polypeptide dynamin ring stabilizer may include
D-amino acid(s) and/or be C-terminal and/or N-terminal protected
against proteolytic digestion (e.g., "pegylated" with
polyethyleneglycol (PEG)). Moreover, peptide or polypeptide dynamin
ring stabilizers can be coupled to a "facilitator moiety" for
facilitating passage or translocation of the peptide/polypeptide
stabilizer across the outer cell/plasma membrane into the cytoplasm
of cells, such as a carrier peptide which has the capacity to
deliver cargo molecules across cell membranes in an
energy-independent manner. Carrier peptides that are known in the
art include penetratin and variants or fragments thereof, human
immunodeficiency virus Tat derived peptide, transportan derived
peptide, signal peptides and fragments thereof which retain the
ability to pass across the outer cellular membrane to effect
delivery of the attached peptide or other agent into the cell.
Rather than a carrier peptide, the facilitator moiety can be a
lipid moiety or other non-peptide moiety which enhances cell
membrane solubility of the dynamin ring stabilizer, such that
passage of the peptide/polypeptide across the cell membrane is
effected. The lipid moiety can for instance be selected from
triglycerides, including mixed triglycerides. Fatty acids and
particularly, saturated C.sub.16-C.sub.20 fatty acids may also be
used (e.g., stearic acid). A peptide or polypeptide dynamin ring
stabilizer can be linked to the facilitator moiety in any
conventionally known manner. For instance, the peptide or
polypeptide can be linked directly to a carrier peptide through an
amino acid linker sequence by a peptide bond or non-peptide
covalent bond using a cross-linking reagent. Moreover, chemical
ligation methods may be used to create a covalent bond between the
carboxy terminal amino acid of the carrier peptide or linker
sequence and the peptide or polypeptide dynamin ring
stabilizer.
The induction of focal adhesions in cells as described herein may
render the cells less able to migrate due to the resulting increase
in cell to cell interactions with neighbouring cells. As such, the
induction of focal adhesions in podocytes or other cells may also
have application in the prophylaxis or treatment of cancer (by
inhibiting cancer cell metastasis), and other diseases or
conditions responsive to the induction of cell focal adhesions.
Any suitable cell can be treated with a dynamin ring stabilizer, or
a prodrug or pharmaceutically acceptable salt thereof, to promote
the formation of and/or for maintenance of dynamin rings in the
cell as described herein. An embodiment of this aspect of the
invention may include selecting the dynamin ring stabilizer (or
prodrug or pharmaceutically acceptable salt thereof) to effect the
formation of the dynamin rings and/or maintenance of the dynamin
rings in the cell. The promotion and/or maintenance of dynamin
rings in, for example, podocytes has particular application in the
prophylaxis or treatment of kidney diseases or conditions
characterized by proteinuria.
The kidney disease or condition characterized by proteinuria for
which the dynamin ring stabilizer is administered in accordance
with an embodiment of the invention can be selected from, but is
not limited to, the group consisting of nephrotic syndrome, chronic
kidney disease, glomerular disease, glomerular dysfunction,
glomerulonephritis including post-infectious glomerulonephritis and
mesangioproliferative glomerulonephritis, nephropathy including
diabetic nephropathy and HIV-associated nephropathy, podocyte
dysfunction including podocyte damage and podocyte injury,
podocytopathies, podocyte foot process effacement, diffuse
mesengial sclerosis, congenital nephrotic syndrome (e.g., of the
Finnish type (CNSF)), Alpor's syndrome and variants (Alport+),
minimal change disease, focal segmental glomerulosclerosis (FSGS),
collapsing glomerulonephropathy, immune and inflammatory
glomerulonephropathies, hypertensive hephrophathy, and age
associated glomerulonephropathy.
The individual treated by a method embodied by the invention can,
for instance, be a member of the bovine, porcine, ovine or equine
families, a laboratory test animal such as a mouse, rat, rabbit,
guinea pig, cat or dog, or a primate or human being. Typically, the
mammal will be a human being.
Suitable pharmaceutically acceptable salts include acid and amino
acid addition salts, base addition salts, esters and amides that
are within a reasonable benefit/risk ratio, pharmacologically
effective and appropriate for contact with animal tissues without
undue toxicity, irritation or allergic response. Representative
acid addition salts include hydrochloride, sulfate, bisulfate,
maleate, fumarate, succinate, tartrate, tosylate, citrate, lactate,
phosphate, oxalate and borate salts. Representative base addition
salts include those derived from ammonium, potassium, sodium and
quaternary ammonium hydroxides. The salts may include alkali metal
and alkali earth cations such a sodium, calcium, magnesium and
potassium, as well as ammonium and amine cations. The provision of
such salts is well known to the skilled addressee. Suitable
pharmaceutical salts are for example exemplified in S. M Berge et
al, J. Pharmaceutical Sciences (1997), 66:1-19, the contents of
which is incorporated herein in its entirety by
cross-reference.
Prodrugs of compounds of the invention include those in which
groups selected from carbonates, carbamates, amides and alkyl
esters have been covalently linked to free amino, amido, hydroxy or
carboxylic groups of the compounds. Suitable prodrugs also include
phosphate derivatives such as acids, salts of acids, or esters,
joined through a phosphorus-oxygen bond to a free hydroxl or other
appropriate group. A prodrug can for example be inactive when
administered but undergo in vivo modification into dynamin ring
stabilizer as a result of cleavage or hydrolysis of bonds or other
form of bond modification post administration. The prodrug form of
the active compound can have greater cell membrane permeability
than the active compound thereby enhancing potency of the active
compound. A prodrug can also be designed to minimise premature in
vivo hydrolysis of the prodrug external of the cell such that the
cell membrane permeability characteristics of the prodrug are
maintained for optimum availability to cells and for systemic use
of the compound.
Esterified prodrugs may for instance be provided by stirring a
compound embodied by the invention with an appropriate anhydride or
acid chloride (in molar excess) in a pyridine/N,N-dimethylformamide
(DMF) solution in the presence of a suitable catalyst such as
dimethylaminopyridine (DMAP). In some cases, the solution may need
to be refluxed to drive the reaction to completion. On completion
of the reaction, the esterified product is purified by either
recrystallization or by chromatography. Representative esters
include C.sub.1-C.sub.7 alkyl, phenyl and phenyl(C.sub.1-6) alkyl
esters. Preferred esters include methyl esters. Examples of
suitable prodrug groups are shown in Table 3.
TABLE-US-00003 TABLE 3 Examples of prodrug groups ##STR00020##
##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025##
##STR00026## ##STR00027## ##STR00028##
For instance, prodrugs of Bis-T-22 and analogues thereof were
developed to increase cell membrane permeability characteristics
and thereby increase potency in cells. A suitable reaction for
providing prodrugs of dimeric tyrphostin compounds is illustrated
in Scheme 3. Bis-T-22 is exemplified as the starting reagent. The
dimeric tyrphostin compound is stirred with appropriate anhydride
or acid chloride (in molar excess) in a
pyridine/N,N-dimethylformamide (DMF) solution in the presence of an
appropriate catalyst such as dimethylaminopyridine (DMAP). In some
cases, the solution may need to be refluxed to drive the reaction
to completion. On completion of the reaction, the esterified
product is purified by either recrystallization or by
chromatography. Particular dimeric benzylidenemalonitrile
tyrphostin prodrugs developed are shown in Table 4 and Table 5.
##STR00029##
TABLE-US-00004 TABLE 4 Prodrugs of bis-tyrphostin (Bis-T-22)
##STR00030## Prodrug R TH-1 CH.sub.3 TH-2 ##STR00031## TH-3
##STR00032## TH-4 ##STR00033## TH-5 ##STR00034## TH-6 ##STR00035##
TH-7 ##STR00036## TH-8 ##STR00037## TH-9 ##STR00038##
TABLE-US-00005 TABLE 5 Further prodrug forms ##STR00039## Prodrug R
Mw Pro-BisT ##STR00040## 616.59 80-1 ##STR00041## 672.68 80-2
##STR00042## 728.78 80-3 ##STR00043## 728.78 80-4 ##STR00044##
841.00 80-5 ##STR00045## 964.04 80-6 ##STR00046## 723.74 81-1
##STR00047## 868.80 81-2 ##STR00048## 928.942
The dynamin ring stabilizer can be administered to an individual in
need of such treatment alone or be co-administered with one or more
other therapeutic compounds or drugs conventionally used for
treating or alleviating symptoms associated with proteinuric kidney
disease. By "co-administered" is meant simultaneous administration
of the drugs in the same formulation or in two different
formulations by the same or different routes, or sequential
administration by the same or different routes, where the drugs act
in overlapping therapeutic windows.
The dynamin ring stabilizer will generally be formulated into a
pharmaceutical composition comprising the stabilizer and a
pharmaceutically acceptable carrier. Injectable solutions will
typically be prepared by incorporating the stabilizer in the
selected carrier prior to sterilizing the solution by filtration.
For oral administration, the dynamin ring stabilizer can be
formulated into any orally acceptable carrier deemed suitable. In
particular, the dynamin ring stabilizer can be formulated with an
inert diluent, an assimilable edible carrier or it may be enclosed
in a hard or soft shell gelatin capsule. Moreover, the dynamin ring
stabilizer can be provided in the form of ingestible tablets,
buccal tablets, troches, capsules, elixirs, suspensions or
syrups.
A pharmaceutical composition as described herein can also
incorporate one or more preservatives such as parabens,
chlorobutanol, phenol, and sorbic acid. In addition, prolonged
absorption of the composition may be brought about by the inclusion
of agents for delaying absorption such as aluminium monosterate.
Tablets, troches, pills, capsules and like can also contain one or
more of the following: a binder such as gum tragacanth, acacia,
corn starch or gelatine, a disintegrating agent such as corn
starch, potato starch or alginic acid, a lubricant, a sweetening
agent such as sucrose, lactose or saccharin, a flavouring agent,
and be provided with an enteric coating to facilitate passage
through the acid environment of the stomach into the small
intestine.
Pharmaceutically acceptable carriers include any suitable
conventionally known physiologically acceptable solvents,
dispersion media, isotonic preparations and solutions including for
instance, physiological saline. Use of such ingredients and media
for pharmaceutically active substances is well known. Except
insofar as any conventional media or agent is incompatible with the
mimetic, use thereof is expressly encompassed. It is particularly
preferred to formulate compositions in dosage unit form for ease of
administration and uniformity of dosage. A dosage unit form as used
herein is to be taken to mean physically discrete units, each
containing a predetermined quantity of the dynamin ring stabilizer
calculated to produce a therapeutic or prophylactic effect. When
the dosage unit form is a capsule, it can contain the active in a
liquid carrier. Various other ingredients may be present as
coatings or to otherwise modify the physical form of the dosage
unit.
Pharmaceutical compositions embodied by the invention will
generally contain at least about 0.1% by weight of the dynamin ring
stabilizer up to about 80% w/w of the composition. The amount of
the dynamin ring stabilizer in the composition will be such that a
suitable effective dosage will be delivered to the individual
taking into account the proposed mode of administration. Preferred
oral compositions will contain between about 0.1 .mu.g and 4000 mg
of the stabilizer.
The dosage of the dynamin ring stabilizer will depend on a number
of factors including whether it is to be administered for
prophylactic or therapeutic use, the disease or condition for which
the active is intended to be administered, the severity of the
condition, the age of the individual, and related factors including
weight and general health of the individual as may be determined in
accordance with accepted medical principles. For instance, a low
dosage may initially be given which is subsequently increased at
each administration following evaluation of the individual's
response. Similarly, frequency of administration can be determined
in the same way that is, by continuously monitoring the
individual's response between each dosage and if necessary,
increasing the frequency of administration or alternatively,
reducing the frequency of administration.
Typically, a dynamin ring stabilizer will be administered in
accordance with a method embodied by the invention at a dosage up
to about 50 mg/kg body weight and preferably, in a range of from
about 1 mg/kg to about 30 mg/kg body weight.
Routes of administration include but are not limited to
intravenously, intraperitonealy, by infusion, orally, rectally, and
by implant. Suitable pharmaceutically acceptable carriers and
formulations useful in compositions of the present invention may
for instance be found in handbooks and texts well known to the
skilled addressee, such as "Remington: The Science and Practice of
Pharmacy (Mack Publishing Co., 1995)", the contents of which is
incorporated herein in its entirety by reference.
The invention will be further described herein after with reference
to non-limiting Examples.
Example 1
Dynamin Assembly Assays
1. Dynamin Self-Assembly Assay--Low Dynamin Concentration
The dynamin self-assembly assay was performed with native dynamin
(40 nM) using the same Hepes column buffer (HCB--20 mM Hepes, 2 mM
EGTA, 1 mM MgCl.sub.2 1 mM PMSF, 1 mM DTT, 20 .mu.g/ml leupeptin,
pH 7.4) as described previously (Warnock et al. 1996). However, all
buffers also included 1% DMSO, which was the vehicle used for the
test dynamin inhibitors. NaCl concentrations were varied from a
stock in 200 mM NaCl. Dynamin was centrifuged at 100,000 g for 20
min, and the supernatants (S) and pellets (P) were collected,
precipitated with trichloracetic acid, solubilised in SDS sample
buffer and separated on SDS gels and dynamin I was detected by
Western blotting using an in house sheep polyclonal .alpha.-dynamin
I antibody.
2. High Dynamin Concentration Self-Assembly Assay--High Dynamin
Concentration
This dynamin self-assembly assay was performed with native dynamin
(5.2 .mu.M) using the Hepes column buffer described above in
Example 1.1, (with the addition of 1% DMSO as described above).
Bis-T effects were measured by pre-incubating the dynamin with the
indicated Bis-T concentrations for 10 min at room temperature
(22.degree. C.). NaCl concentrations were varied from a stock in
200 mM NaCl. After the incubation the tubes were transferred to a
TLA120.2 rotor (Beckman) and centrifuged in a tabletop
ultracentrifuge (Optima TLX, Beckman) at 100,000 g for 20 min, and
the supernatants (S) and pellets (P) were collected, precipitated
with trichloracetic acid, solubilised in SDS sample buffer and
separated on SDS gels.
Example 2
Dynamin Ring Stabilizers are a Subgroup of Dynamin Inhibitors
Among the various classes of dynamin inhibitors are subsets of
dynamin ring stabilizers. Bis-T dimeric benzylidenemalonitrile
tyrphostins potently inhibit helical dynamin GTPase activity and
can stimulate the basal activity via promotion of ring formation in
the absence of a template for dynamin helical assembly. FIG. 1
shows the in vitro GTPase activity of purified sheep brain dynamin
(mostly this is dynamin I) at increasing dynamin concentrations,
below those required for ring assembly. The filled symbols (solid
lines) show that dynamin is stimulated by liposomes at all dynamin
concentrations tested (forming a helix).
2-Cyano-N-{3-[2-cyano-3-(3,4,5-trihydroxyphenyl)-acryloylamino]-p-
ropyl}-3-(3,4,5-trihydroxyphenyl)-acrylamide (Bis-T-23) at 5 .mu.M
potently inhibits dynamin at all points (however at very high
dynamin (400 nM), Bis-T-23 fails to inhibit, potentially because of
the high dynamin concentration).
In contrast, in the absence of liposomes (where dynamin is unable
to form helices) the results are strikingly opposite (FIG. 1, open
symbols, dotted lines). Using dynamin concentrations below those
necessary to form rings, Bis-T-23 potently stimulates dynamin
activity. In contrast to helical dynamin which is inhibited at all
dynamin concentrations, in the absence of liposomes Bis-T-23 does
not activate or inhibit dynamin at low dynamin concentrations, but
stimulates only at 100 nM or higher dynamin concentration (FIG. 1).
This indicates that Bis-T-23 is neither an activator nor inhibitor
of the basal GTPase activity of dynamin, but is affecting ring
dynamin.
The IC.sub.50 for inhibition of PS-stimulated helical dynamin in
this experiment is study 500 nM (FIG. 2). The concentration of
Bis-T-23 causing 50% activation of basal dynamin activity
(EC.sub.50) was strikingly about the same value. This suggests the
mechanism of ring stabilization and of inhibition may be related
for this series of compounds.
Next, a time course experiment of Bis-T-23 stimulation of dynamin
GTPase (FIG. 3) was conducted. Using high dynamin concentrations
(800 nM) and medium NaCl concentrations (30 mM), dynamin basal
GTPase activity was linearly increased with time, since it did not
assemble as rings in these conditions. In the presence of 5 .mu.M
Bis-T-23 dynamin activity was greatly increased after an initial
"lag phase" of about 4 minutes. The lag phase is thought to
indicate the time required for dynamin to assemble as rings. The
results show that Bis-T-23 does not accelerate initial rates of
dynamin ring assembly, suggesting it instead inhibits ring
disassembly.
When a series of potent iminodyn dynamin inhibitors were tested for
ring stabilizer activity, a subset were found to be potent
activators of basal dynamin GTPase activity (ie in the absence of
PS; FIG. 4). Iminodyns-17, 22 and 23 had stimulation values
(EC.sub.50) from 1-20 .mu.M, indicative of ring stabilizer
activity.
There was little correlation between IC.sub.50 for PS-stimulated
dynamin and EC.sub.50 for dynamin in the absence of PS. Therefore,
only a subset of dynamin inhibitors are ring stabilizers and ring
stabilizers cannot be recognised as simply potent inhibitors of
PS-stimulated dynamin GTPase activity (Table 2). For example,
iminodyns 17 and 22 are equipotent dynamin inhibitors yet are 30
fold different in ring stabilizer activity. Likewise, iminodyns 20
and 21 are similarly potent dynamin inhibitors yet iminodyn 20
exhibits no ring stabilizer activity (Table 2). Thus ring
stabilizer activity can be identified by the ability to stimulate
the activity of dynamin protein, in the absence of other
stimulatory factors (such as PS liposomes, microtubules or
nanotubes).
To verify that ring stabilizers require dynamin oligomerisation, a
recombinant form of dynamin, called GG-2, which is dimeric yet is
unable to self-associate into higher order oligomers (Chappie et al
2009) was tested. Ring stabilizers are unable to stimulate the
basal activity of such constructs (FIG. 5).
The dynamin concentration-dependence of the activation effect of
Bis-T-23 suggested it may be altering the in vitro formation of
dynamin rings. A block in ring disassembly should be manifested as
an accumulation of rings in vitro. Dynamin self-assembles into
rings in the absence of any cofactors when the buffer ionic
strength is decreased (Song et al. 2004). To determine whether
Bis-T compounds regulate dynamin-dynamin interactions a
well-established method of high speed centrifugation in decreasing
amounts of NaCl to collect rings in the pellet (Warnock et al.
1996) was used. Higher concentrations of dynamin (520 nM) and
dynamin was visualized by Coomassie staining on SDS gels (FIG. 6).
As reported by others, dynamin is in the supernatant at .gtoreq.40
mM NaCl, but self-assembled at .ltoreq.20 mM NaCl and is found in
the pellet. Bis-T-22 at 100 .mu.M increased ring assembly at the
intermediate salt concentrations (40, 60 nM) prior to its
spontaneous assembly at lower salt. At a fixed 40 mM NaCl, Bis-T-22
promoted all dynamin to form rings at 300 .mu.M concentrations.
Therefore, under these conditions Bis-T-22 stabilized/promoted
dynamin I self-assembly. Thus, it was concluded that there is a
direct effect on dynamin-dynamin interactions, with the compound
appearing to promote dynamin ring assembly and GTPase activity in
vitro by virtue of preventing ring disassembly. Hence, GTPase
active rings accumulate.
To directly demonstrate whether dynamin ring stabilizers prevent or
retard the disassembly of dynamin rings a well established
centrifugation assay was employed. Briefly, dynamin I was
preassembled as a helix in the presence of PS liposomes for 30 min,
after which the dynamin ring stabilizer Bis-T-23 or non-dynamin
ring stabilizer dynole 34-2 (see Example 4) were added. The samples
were then centrigued in a microfuge for 10 min to collect
supernatans (Sup) or pellets. In this assay, helical dynamin is
found primarily in the pellet. However, the addition of either NaCl
(150 mM) or Mg/GTP (1 mM) is known to disassemble dynamin and it is
found primarily in the supernatant (see FIG. 7). Bis-T-23 was found
to reduce the disassembly of helical dynamin, while dynole 34-2 did
not (FIG. 7). This shows that the mechanism whereby Bis-T acts as a
dynamin ring stabilizer is, or includes, retarding dynamin ring or
helix disassembly. This unique mechanism of action explains why
dynamin ring stabilizers can stimulate basal GTPase while
inhibiting helical dynamin GTPase activity at the same time.
Example 3
Bis-T-23 Produces Inflexible Dynamin Rings In Vitro
A characteristic of helical dynamin is that the helix is a highly
flexible structure. Upon GTP hydrolysis it is able to rapidly
reduce its diameter (constriction) while also expanding in length
(helical expansion) (Stowell et al. 1999; Chen et al. 2004; Roux et
al. 2006). This is thought to be a potential mechanism utilized for
the fission of the necks of newly budded endocytic vesicles in
cells (Roux et al. 2006). The highly flexible nature of helical
dynamin was confirmed by EM analysis of dynamin bound to
phosphatidylserine liposomes in the absence of GTP or GDP (FIG. 8).
Dynamin mixed with PS liposomes formed a helix with specific
characteristics (FIGS. 8A and C): the individual loops of the helix
were highly varied in diameter, formed at different angles relative
to the underlying lipid, and the spacing between loops was highly
variable. In contrast, 5 .mu.M Bis-T-23 drastically altered the
helical shape (FIGS. 8B and D). Notably the individual loops of the
helix were highly uniform in diameter, formed at constant angles
relative to the underlying lipid, and the spacing between loops was
highly constant. Bis-T-23 bound dynamin appeared similar to
previous reports of GTP.gamma.S-bound dynamin on lipid nanotubes
(Stowell et al. 1999). This indicates that Bis-T-23 can lock
dynamin loops into an inflexible and uniform diameter, akin to the
GTP-bound state, by preventing ring disassembly. The effect is
particularly noticeable at the ends of each helical tube, which are
"tapered" in the absence of Bis-T-23, yet "blunt" in its presence
(FIG. 8C-D), demonstrating the inflexibility of Bis-T-23 affected
loops.
The electron microscope (EM) results revealed a common mechanism
for both inhibition and activation of dynamin GTPase. It was
concluded that the super-elevated GTPase activity of helical
dynamin is inhibited by Bis-T-23 because the drug renders the loops
of the helix inflexible. In the absence of PS liposomes, the
activity of the individual dynamin rings induced by the presence of
Bis-T-23 is stimulated due to the same accumulation of uniformly
sized inflexible rings. Thus, it was concluded that Bis-T-23
prevents the dynamin rapid disassembly that would normally be
driven by GTP hydrolysis, which drives dynamin disassembly in
vitro.
Next, the effect of a ring stabilizer Bis-T-23 on the formation of
dynamin rings in the absence of a template like PS liposomes was
examined. At high concentrations in vitro, dynamin is well known to
self-assemble into rings that are detectable by EM (Hinshaw and
Schmid, 1995). Such self assembly requires high dynamin
concentrations typically in the order of 500-1000 nM and is not
observed at lower dynamin concentrations. Specifically, the effect
of Bis-T-23 (5.4 .mu.M) on 200 nM dynamin in the absence of
template was tested, which is well below the concentration
threshold for self-assembly. As expected, dynamin did not
self-assemble to appreciable levels at this concentration (FIG.
9A). However in the presence of Bis-T-23, dynamin showed an
unanticipated massive increase in self assembly (FIG. 9A). High
power images confirmed that the electron dense structures are bona
fide dynamin rings (FIG. 9B). Quantitative analysis of about 2000
dynamin rings showed a massive increase in ring formation induced
by Bis-T-23 (FIG. 9C). These results confirm that Bis-T-23 promotes
the accumulation of dynamin rings by either inducing their
formation or by preventing their disassembly. The findings with
basal dynamin are completely unexpected for a dynamin inhibitor and
are indicative of the ring stabilizer activity.
Example 4
Bis-T-22 Produces Inflexible Dynamin Rings in Cells
This Example shows that a dynamin ring stabilizer is able to
promote dynamin ring formation in cells and prevent or retard their
disassembly. When vesicles are endocytosed via the
clathrin-dependent pathway, they are well known to be internalised
as omega shaped figures close to the plasma membrane with partially
constricted and short necks. These can be detected by transmission
electron microscopy (EM) at a low frequency. Treatment of cells for
10-30 minutes with a classical dynamin inhibitor such as dynasore
causes a massive accumulation of clathrin coated pits at the plasma
membrane of cells without promotion of dynamin rings (Macia et al,
2006). Other dynamin inhibitors like MiTMAB (Quan et al, 2007) or
dynole 34-2 do not induce any accumulation of coated pits,
presumably because they at least partly act at the lipid surface to
prevent their initial formation and are inhibitors without ring
stabilizer characteristics. In contrast to these observations, a
dynamin ring stabilizer causes accumulation of clathrin coated pits
in cells with two distinctive features: the vesicle necks are
highly elongated and are encircled by electron dense rings. Human
lymphoblasts (which express dynamin II) were treated with dynasore,
MiTMAB (not shown) or Bis-T-22 for 30 min and prepared for EM
analysis. While dynasore and MiTMAB produced the expected outcomes
reported previously, Bis-T-22 elicited a massive accumulation in
all cells of clathrin coated pits with highly elongated necks and
which were encircled by rings or spirals (FIG. 10A). Similarly,
when rat brain synaptosomes (isolated nerve terminals, which
primarily express dynamin I) were treated with Bis-T-22 for 30 min
and depolarised with KCl to evoke endocytosis, trapped endocytic
pits were detected that were encircled by a single electron dense
ring (FIG. 10B lower 3 panels). No rings or trapped vesicles were
detected when the synaptosomes were unstimulated (FIG. 10B top).
This mimics the in vitro observations on purified dynamin I
described earlier which forms single rings (FIG. 9).
These observations illustrate that ring stabilizers have the
ability to promote and stabilise rings at sites of trapped
endocytosis in distinct cellular types. This characteristic is not
found with dynamin inhibitors that are not dynamin ring
stabilizers, supporting that dynamin ring stabilization is a novel
action of ring stabilizer compounds which can occur in the context
of live cells and is not restricted to in vitro conditions with the
purified protein.
Example 5
Not all Dynamin Inhibitors are Dynamin Ring Stabilizers
A new series of potent dynamin GTPase inhibitors based on the
structure of dynasore (Macia et al. 2006) was designed. These
compounds were called dyngo's. The most active dyngo analogue is
dyngo-4a, with an IC.sub.50 for PS-stimulated dynamin of 300 nM, in
comparison with dynasore (dyngo-7a) IC.sub.50 of 14 .mu.M. The
structure of the dyngos resembles a monomeric form of the Bis-T and
also monomeric tyrphostins. However, it was found that the dyngos,
and especially dynasore, strongly bind to the detergent Tween-80
which is a normal component of assays to screen for dynamin
inhibitors (Quan and Robinson 2005). Upon performing the basal
GTPase assay in the present studies in the absence of Tween-80, it
was found that both the dyngos and dynasore stimulated basal
dynamin GTPase activity to similar extents as Bis-T-23 (FIG. 10A).
This shows that basal dynamin GTPase activation (i.e., dynamin ring
activation) is not restricted to Bis-T tyrphostins nor
iminodyns.
Another potent dynamin inhibitor series in a novel chemical class
are the "dynole" series of compounds, which are indole-based
inhibitors as described in International Patent Application No.
PCT/IB2008/002387 (WO 2009/034464) (see also Hill et al. 2009). The
most potent dynole developed to date is dynole 34-2
(2-cyano-3-(1-(2-(dimethylamino)-ethyl)-1H-indol-3-yl)-N-octylacrylamide)
with an IC.sub.50 for PS-stimulated dynamin of 1 .mu.M. Dynole 34-2
failed to stimulate the basal activity of dynamin (FIG. 11A).
Hence, it is not a dynamin ring disassembly inhibitor (i.e., not a
dynamin ring stabilizer).
Next, a range of potent Bis-T analogues were tested in the standard
GTPase assay employed in the presence of Tween-80. Four of the most
potent dynamin inhibitors (Hill et al. 2005) were also found to be
dynamin ring stabilizers since they increased basal activity (FIG.
11B). Dyngo-4a was also effective, although dynasore failed to
stimulate basal GTPase activity under these conditions (due to its
non-specific Tween-80 binding). Importantly, several dynamin
inhibitors in the MiTMAB series (which target the PH domain, Quan
et al 2007) or dynole series (which target an allosteric site in
the GTPase domain, Hill et al. 2009) failed to stimulate basal
dynamin GTPase activity (FIG. 11B). Therefore, dynamin inhibitors
with distinct mechanisms of action on dynamin do not all increase
the basal dynamin GTPase activity (i.e., they are not all dynamin
ring stabilizers). This indicates that ring stabilizers represent a
distinct class of dynamin modulator, not specifically connected to
their ability or not to inhibit template-stimulated dynamin GTPase
activity in vitro.
In summary, a variety of dynamin ring stabilizers from a number of
distinct chemical classes were identified herein by their ability
to stimulate the basal activity of full length dynamin. The
mechanism of stimulation was explained since these compounds
specifically stabilize dynamin self-assembly into single rings
(thereby stimulating the basal rate of GTP hydrolysis), most likely
by inhibiting dynamin disassembly. Not all the dynamin ring
stabilizers were potent inhibitors of helical dynamin GTPase
activity. Importantly, dynamin self-assembly into rings has a
specific and selective effect on the actin cytoskeleton (see
below), and these compounds are able to stabilize or prolong the
function of dynamin rings in the actin cytoskeleton.
Example 6
Actin Stimulates Dynamin Ring Assembly In Vitro
In the present study, it was found that the formation of rings by
dynamin is essential for increasing the actin cytoskeleton in
podocytes, and direct interactions between dynamin and filamentous
actin (F-actin) were identified. In particular, an unrecognized
actin binding site in dynamin was identified that binds along actin
filaments and aligns them into bundles. F-actin, and in particular,
short filaments capped on their barbed ends by gelsolin (Gsn),
promote dynamin ring formation and stimulate its GTPase activity.
This interaction, in turn, dissociates gelsolin from the barbed
filament ends and promotes filament elongation. The reciprocal
interplay between dynamin and Gsn-capped filaments can thus
influence the architecture and dynamics of actin. Dynamin mutants
defective in actin-binding in vitro have impaired oligomerization
in cells and reduce actin stress fiber assembly, and altered
cortical actin cytoskeletal behavior in cultured podocytes. In
contrast, a dynamin mutant with increased actin affinity has an
increased propensity to oligomerize in the cytoplasm and stimulates
stress fiber assembly in the perinuclear region of the cell. These
findings suggest a complex interplay between dynamin's GTPase cycle
and the global organization of the actin cytoskeleton in
podocytes.
Example 7
Dynamin Binds F-Actin
To determine whether dynamin affects the actin cytoskeleton, the
question of whether dynamin might directly bind filamentous actin
(F-actin) was tested. In particular, an actin co-sedimentation
assay was performed in which F-actin sediments under high-speed
centrifugation. If dynamin interacts with F-actin it would be
expected to co-sediment, and thus be present in the pellet
fraction. In the presence of F-actin, but not in its absence, the
majority of dynamin was found in the pellet (FIG. 12, lanes 2 and
14). Dynamin-actin interactions did not require the presence of
GTP, indicating they did not require dynamin oligomerization.
Moreover, dynamin binding to F-actin was observed in the presence
of GTP.gamma.S (FIG. 12, lane 6), indicating dynamin
oligomerization was not inhibitory for binding. The Kd of dynamin
for actin was approximately 0.4 .mu.M, which is similar to the
affinities of other actin binding proteins such as .alpha.-actinin
4 for actin (Weins et al. 2005).
Example 8
The Actin Binding Domain of Dynamin is in the Dynamin Middle
Domain
Next, the actin binding site was mapped to a region between amino
acids 399 and 444 amino acids of dynamin II (dyn2) (FIG. 13). As
predicted for an actin binding domain (Van et al. 1996), this
region contains several positively charged amino acids conserved in
all dynamin gene products from yeast to mammals. It is also
alternatively spliced within different mammalian dynamin isoforms
(called variant a and b in dynamins I and II) (see FIG. 13, SEQ ID
No. 1-4). Site-directed mutagenesis was performed on conserved,
charged residues within this actin binding domain to generate the
putative `loss of function` mutants, dynK/E (SEQ ID No. 9) and
dynK/A (SEQ ID No. 10), and a putative `gain of function` mutant,
dynE/K (SEQ ID No. 11) (FIG. 13). The affinities of dynK/E and
dynK/A for actin were found to be reduced (Kd of 1.7 and 2.8 .mu.M,
respectively), whereas the affinity of dynE/K for actin was
increased (Kd=0.03 .mu.M) (FIG. 14). All three mutant proteins had
wild-type kinetic properties with respect to the basal and
stimulated rate of GTP hydrolysis, indicating proper folding.
Example 9
Dynamin-Actin Interactions are Involved in the Actin Cytoskeleton
of Podocytes
To determine the role of dynamin-actin interactions in actin
organization, the consequences for podocyte morphology of
expressing dynamin mutants with altered affinity for F-actin was
examined. In fully differentiated mouse podocytes, the actin
cytoskeleton is organized in parallel bundles of actin-myosin
contractile stress fibers in the cell body and a cortical network
of short, branched actin filaments located beneath the plasma
membrane that drives formation of lamellipodia and filopodia.
Expression of dynK44A, a dynamin mutant that cannot bind GTP,
abolished stress fibers within the cell body and generated a thick,
hyper-bundled actin network in the vicinity of the plasma membrane,
causing a polygonal cellular shape (Sever et al. 2007). In the
present study it was found that expression of dynK44A abolished
formation of lamellipodia and filopodia (FIG. 15B). Expression of
the `loss of function` mutant dynK/E resulted in reduction of
stress fibers and dramatic changes in cell shape (FIG. 15C). In
contrast, expression of the `gain of function` dynE/K caused a
clear increase in stress fibers within the cell body (FIG. 15D). A
similar increase in stress fibers was observed in cells expressing
dynR725A (FIG. 15E), the mutant that lives longer in the assembled
GTP-bound state.
Example 10
Dynamin Rings Cross-Link F-Actin into Tight Bundles
To evaluate the effects of dynamin on the structure of actin
filaments, F-actin was examined using negative staining and
electron microcopy (EM). Dynamin oligomerized into rings by the
addition of GTP.gamma.S (a non-hydrolysable GTP analog that
promotes its ring formation) and crosslinked actin filaments into
tight bundles (FIG. 16). The filament-to-filament spacing in these
bundles was 17-20 nm, less than the diameter of a dynamin ring
(.about.50 nm). Thus, these findings suggest that in the absence of
other actin binding proteins dynamin rings can form actin bundles
composed of parallel filaments with well-defined spacing. Such
parallel actin filaments occur in stress fibers and filopodia. This
is believed to be the first report of a biological function for the
ring form of dynamin.
Example 11
Dynamin Rings Displace Gelsolin from Barbed Filament Ends
Next, the question of whether dynamin can expose the barbed ends of
gelsolin (Gsn) capped F-actin was tested. Actin was polymerized in
the presence of gelsolin at the indicated ratios (1G:A200 or
1G:A1000) (FIG. 17). Under these conditions, gelsolin caps >99%
of the barbed end. The filament length is defined by the Gsn:actin
ratio (.about.0.5 .mu.m or 2.7 .mu.m, respectively) and the extent
of actin polymerization is controlled by the critical concentration
of pointed filament ends which is .about.0.6 .mu.M. The actin
solution was then diluted to 0.33 .mu.M in the presence or absence
of dynamin. Under these conditions, actin can only repolymerize if
barbed ends become available. Addition of dynamin induced actin
polymerization, but only in the presence of GTP.gamma.S (FIG. 17,
compare the diamonds and circles with the squares). This is
consistent with dynamin rings having the capacity to displace
gelsolin from the barbed filament ends either directly or by
altering F-actin structure at its barbed ends. Overall, the data
establish an interconnection between F-actin dynamics and dynamin
oligomerization. In particular, short, gelsolin capped actin
filaments promote dynamin ring formation, which in turn dissociates
gelsolin from the barbed ends and allows filament elongation.
Example 12
Bis-T-23 Stimulates Formation of Dynamin Rings
Both dynR725A and dynE/K are predicted to live longer in dynamin
ring formation (Sever et al. 2007). DynR725A was previously
reported to rescue proteinuria in an LPS model by oligomerizing
into the rings and thus avoiding cleavage by the protease cathepsin
L (CatL) (Sever et al. 2007). The expression of dynR725A is
sufficient to reduce proteinuria in a mouse model of nephrotic
syndrome (Sever et al. 2007).
The question of whether Bis-T-22 and Bis-T-23 might activate wild
type ring dynamin and phenocopy the dynR725A phenotype was tested.
Bis-T-23 increased the rate of basal dynamin GTP hydrolysis to a
similar level to addition of Gsn-F-actin complexes (FIG. 18). Thus,
both Bis-T-23 and Gsn-F-actin complexes promote dynamin
oligomerization into rings. When both reagents were simultaneously
present, there was additional stimulation of dynamin's GTPase
activity (FIG. 18). Together, these data suggest that Bis-T-23 does
not compete for F-actin binding by dynamin, and that both
components act additively or synergistically with respect to
dynamin oligomerization.
Example 13
Bis-T-23 Stimulates Focal Adhesions and Stress Fibers in Cultured
Podocytes
To determine if the in vitro action of the dynamin ring stabilizers
also occurs in cells, the effect of Bis-T-23 on the actin
cytoskeleton in cultured mouse podocytes was tested (FIG. 19). 10
min after addition of Bis-T-23 there was a dramatic increase in
well-defined stress fibers (FIG. 19B-D). This was associated with a
dramatic increase in the number of focal adhesions (paxillin
staining in FIG. 19B). While Bis-T-23 inhibits endocytosis (not
shown), the actin cytoskeleton is profoundly different compared to
cells expressing dynK44A (loss of stress fibers and increase in
hyper bundled actin filaments). Thus, the observed increase in
stress fibers is unlikely due to inhibition of endocytosis.
These results show that dynamin rings have a role in the formation
of stress fibers and focal adhesions in cultured podocytes, and
that dynamin ring stabilizer molecules such as Bis-T may inhibit or
reverse proteinuria by restoring functional FP due to actin
reorganization.
Example 14
Inhibition of Proteinuria in Animal Models
To demonstrate the effectiveness of a ring stabilizer in vivo, two
different mouse models of kidney disease were utilised, a genetic
model and an acute toxicity model. Mice expressing a `gain of
function` mutation in the ACTN4 gene encoding for .alpha.-actinin 4
has been fully characterized (Kaplan et al. 2000; Henderson et al.
2008; Yao et al. 2004). These animals develop FP effacement and
proteinuria due to "hyper bundling" activity of .alpha.-actinin 4
mutant that induces aggregation of stress fibers. Proteinuria
develops at 4-6 weeks of age. The animals used in the present study
were obtained from Dr. Martin Pollak, Brigham and Women's Hospital,
Boston, Mass., USA. Proteinuric phenotype was confirmed by
determination of urinary albumin and creatinine using mouse
Albumin-specific ELISA and Creatinine Companion assay kits (Exocell
and Bethyl Laboratories) following the manufacturer's protocols.
The control animal was a litermate. Bis-T-23 was dissolved in 100%
DMSO to make a 10 .mu.g/.mu.l stock solution, of which 5 .mu.l was
diluted in 200 .mu.l of 1.times.PBS, and this was injected
intraperitoneally (IP) into the test animal (166 .mu.g/100 g body
weight) at time 0 hr. Protein levels in the urine were measured
every 2 hours post injection. The results are shown in FIG. 20. As
can be seen, a decrease in proteinuria to wild-type levels was
obtained up to 6 hours after administration of the Bis-T-23.
A second model of acute proteinuria was then employed to test the
ability of a dynamin ring stabilizer to ameliorate proteinuria in a
reversible model of proteinuric kidney disease. LPS-induced
proteinuria was utilized as previously described (Sever et al.,
2007). Briefly, four week old female BALB/c mice were injected
twice intraperitoneally (IP) with 200 .mu.g of ultrapure LPS
diluted in phosphate-buffered saline at a concentration of 1 mg/ml.
Proteinuria developed within 48 h (FIG. 20, 2.times.LPS). Bis-T-23
was dissolved in DMSO to the appropriate concentrations and 300
.mu.g per 100 g body weight was delivered by IP injection, and DMSO
was used as a vehicle control. The level of protein in the urine
was determined by using mouse Albumin-specific ELISA kit according
to manufacturer's protocol (Bethyl Laboratories). Administration of
the Bis-T-23 partially rescued proteinuria from 2-6 h after
injection (FIG. 21). Its ability to rescue proteinuria was reduced
8 h after injection. These results show that a ring stabilizer can
ameliorate proteinuria in a mouse model of proteinuric kidney
disease.
Although a number of embodiments have been described, it will be
appreciated by persons skilled in the art that numerous variations
and/or modifications may be made without departing from the spirit
or scope of the invention as broadly described. The present
embodiments are, therefore, to be considered in all respects as
illustrative and not restrictive.
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SEQUENCE LISTINGS
1
11146PRTMus musculus 1Arg Thr Gly Leu Phe Thr Pro Asp Leu Ala Phe
Glu Ala Ile Val Lys1 5 10 15Lys Gln Val Val Lys Leu Lys Glu Pro Cys
Leu Lys Cys Val Asp Leu 20 25 30Val Ile Gln Glu Leu Ile Ser Thr Val
Arg Gln Cys Thr Ser 35 40 45246PRTMus musculus 2Arg Thr Gly Leu Phe
Thr Pro Asp Met Ala Phe Glu Ala Ile Val Lys1 5 10 15Lys Gln Leu Val
Lys Leu Lys Glu Pro Ser Leu Lys Cys Val Asp Leu 20 25 30Val Val Ser
Glu Leu Ala Thr Val Ile Lys Lys Cys Ala Glu 35 40 45346PRTMus
musculus 3Arg Thr Gly Leu Phe Thr Pro Asp Leu Ala Phe Glu Ala Ile
Val Lys1 5 10 15Lys Gln Val Gln Lys Leu Lys Glu Pro Ser Ile Lys Cys
Val Asp Met 20 25 30Val Val Ser Glu Leu Thr Ser Thr Ile Arg Lys Cys
Ser Glu 35 40 45446PRTMus musculus 4Arg Thr Gly Leu Phe Thr Pro Asp
Met Ala Phe Glu Thr Ile Val Lys1 5 10 15Lys Gln Val Lys Lys Ile Arg
Glu Pro Cys Leu Lys Cys Val Asp Met 20 25 30Val Ile Ser Glu Leu Ile
Ser Thr Val Arg Gln Cys Thr Lys 35 40 45546PRTDrosophila
melanogaster 5Arg Val Gly Leu Phe Thr Pro Asp Met Ala Phe Glu Ala
Ile Val Lys1 5 10 15Arg Gln Ile Ala Leu Leu Lys Glu Pro Val Ile Lys
Cys Val Asp Leu 20 25 30Val Val Gln Glu Leu Ser Val Val Val Arg Met
Cys Thr Ala 35 40 45646PRTCaenorhabditis elegans 6Arg Val Gly Leu
Phe Thr Pro Asp Met Ala Phe Glu Ala Ile Ala Lys1 5 10 15Lys Gln Ile
Thr Arg Leu Lys Glu Pro Ser Leu Lys Cys Val Asp Leu 20 25 30Val Val
Asn Glu Leu Ala Asn Val Ile Arg Gln Cys Ala Asp 35 40
45746PRTSaccharomyces cerevisiae 7Ala Pro Ser Leu Phe Val Gly Thr
Glu Ala Phe Glu Val Leu Val Lys1 5 10 15Gln Gln Ile Arg Arg Phe Glu
Glu Pro Ser Leu Arg Leu Val Thr Leu 20 25 30Val Phe Asp Glu Leu Val
Arg Met Leu Lys Gln Ile Ile Ser 35 40 45846PRTMus musculus 8Arg Pro
Thr Leu Phe Val Pro Glu Leu Ala Phe Asp Leu Leu Val Lys1 5 10 15Pro
Gln Ile Lys Leu Leu Leu Glu Pro Ser Gln Arg Cys Val Glu Leu 20 25
30Val Tyr Glu Glu Leu Met Lys Ile Cys His Lys Cys Gly Ser 35 40
45946PRTMus musculus 9Arg Thr Gly Leu Phe Thr Pro Asp Leu Ala Phe
Glu Ala Ile Val Glu1 5 10 15Glu Gln Val Val Glu Leu Glu Glu Pro Cys
Leu Glu Cys Val Asp Leu 20 25 30Val Ile Gln Glu Leu Ile Ser Thr Val
Arg Gln Cys Thr Ser 35 40 451046PRTMus musculus 10Arg Thr Gly Leu
Phe Thr Pro Asp Leu Ala Phe Glu Ala Ile Val Ala1 5 10 15Ala Gln Val
Val Ala Leu Ala Glu Pro Cys Leu Ala Cys Val Asp Leu 20 25 30Val Ile
Gln Glu Leu Ile Ser Thr Val Arg Gln Cys Thr Ser 35 40 451146PRTMus
musculus 11Arg Thr Gly Leu Phe Thr Pro Asp Leu Ala Phe Glu Ala Ile
Val Lys1 5 10 15Lys Gln Val Val Lys Leu Lys Lys Pro Cys Leu Lys Cys
Val Asp Leu 20 25 30Val Ile Gln Lys Leu Ile Ser Thr Val Arg Gln Cys
Thr Ser 35 40 45
* * * * *
References